: A long-standing goal in biology is to define the function of each gene encoded by the human genome. Seminal work in yeast established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Recent efforts in human cells focusing on small gene sets have underscored the utility of this approach, but the feasibility of generating large- scale maps similar to approaches in yeast remains to be determined. We have recently developed a new unpublished CRISPR interference platform for large-scale quantitative mapping of human GIs. Our genetic interaction (GI) maps systematically perturbed 222,784 gene pairs in each of two hematopoietic cancer cell lines. These maps functionally cluster related genes and assign function to poorly characterized genes, including TMEM261, a new electron transport chain Complex 1 gene, demonstrating that GI maps reveal fundamental new biology. Specific GIs pinpoint unexpected relationships between cellular pathways, exemplified by a cholesterol biosynthesis intermediate that links to DNA repair. Additionally, we demonstrate that specific buffering and synthetic sick/lethal GIs inform therapeutic strategies for human diseases. More generally, these GI maps elucidate the nature and frequency of GIs in human cells. Our work establishes GI mapping as a high-resolution tool for unbiased dissection of gene function and serves as a blueprint for mapping the genetic landscape of human cells. Here, I propose to apply this new method to create a large-scale GI map of thousands of gene products localized to the human nucleus. Biologists have defined the sequence of the genome, catalogued many functional DNA elements and explored in detail the function of specific gene products. However, for many protein coding and non-coding genes localized to the nucleus, we have little understanding of their molecular function, let alone how the activity of individual gene products is integrated into higher order functional units fundamental to epigenetics, transcription, splicing and DNA repair. This GI map will systematically and in an unbiased manner elucidate how sets of genes encode the biology of nuclear protein complexes, pathways and ultra-structures and will also inform our understanding of specific disease processes in human cells. The results of the proposed research will serve as a fundamental resource for a broad community of biomedical scientists and greatly inform our understanding of human biology and disease.